Age‐ and endometrial microbiota‐related delay in development of endometrial receptivity

Abstract Purpose We evaluated factors affecting the development of endometrial receptivity according to age and changes in the endometrial microbiota. Methods We recruited patients with infertility who underwent transcriptomic analyses of endometrial receptivity and the endometrial microbiome prior to frozen embryo transfer. An endometrial biopsy was performed 108 h after initial progesterone administration. Results In 185 tests from 185 eligible patients, the results of endometrial receptivity analysis were receptive in 111 (60.0%) patients and pre‐receptive in 74 (40.0%) patients. Compared with receptive patients, pre‐receptive patients had significantly older ages (36.0 ± 0.5 vs. 38.2 ± 0.5, p = 0.0021), a smaller proportion of normal Lactobacillus‐dominant microbiota (27.9% vs. 12.2%), and a greater proportion of microbiota with ultralow biomass (22.5% vs. 41.9%) (p = 0.0074). Patient age (adjusted odds ratio: 1.08, 95% confidence interval: 1.01–1.16, p = 0.0351) and a microbiome with ultralow biomass (adjusted odds ratio: 3.82, 95% confidence interval: 1.49–9.82, p = 0.0039) were independent predictive factors for pre‐receptive endometrium. Conclusions Older age was accompanied by a decrease in Lactobacillus‐dominant microbiota; aging and endometrial microbiota with ultralow biomass were significantly associated with pre‐receptive endometrium. Our findings suggest that the quantity (rather than proportion) of Lactobacillus in the endometrium is important in the development of endometrial receptivity.


| INTRODUC TI ON
Endometrial receptivity for embryos is an important but minimally investigated topic in assisted reproductive technology (ART). With advancements in genome technology, the endometrial receptivity array has become established as a novel diagnostic tool for measuring potential transcriptomic profiles related to embryo implantation; 1 this approach is more accurate and consistent than conventional analyses using biochemical markers or histopathology methods. The upregulation of molecules related to immune modulation, adhesion, or angiogenesis is essential for endometrial receptivity, and progesterone may be involved in this process. 2 A recently created transcriptomic atlas of the human endometrium at single-cell resolution clearly demonstrated that the window of implantation (WOI) opens with abrupt and discontinuous transcriptomic activation of the epithelia and stromal fibroblasts. 3 Endometrial receptivity analysis (ERA, ©Igenomix) is the first commercial test available for clinical analysis of the expression patterns of 248 genes to identify the receptivity status (i.e., receptive or non-receptive) of the endometrium and determine WOI displacement in a particular patient. 1,4 Patients with receptive results according to ERA can undergo standard frozen embryo transfer (FET) in a subsequent cycle; in patients with non-receptive results, adjustment of embryo transfer timing (so-called personalized embryo transfer, pET) is recommended to improve ART outcomes. 2,5,6 A recent systematic review and meta-analysis 7 demonstrated that onethird of infertile women had a displaced WOI, and pET may facilitate implantation in patients with recurrent implantation failure caused by non-receptive endometrium.
There is a growing body of evidence that microbiota, which are specific microbe populations that symbiotically inhabit the host, communicate with the host through humoral signaling molecules, thus influencing overall health status. 8 Recent studies have suggested connections between microbiota and female reproductive tract disorders, such as bacterial vaginosis, cervical and endometrial cancer, polycystic ovary syndrome, postmenopausal syndrome, endometriosis, endometritis, uterine fibroids, and infertility. [9][10][11] Throughout the female reproductive tract, Lactobacillus spp. are the most frequently identified bacteria. Analyses of the V3-V4-V6 regions in the 16S rRNA gene via next-generation sequencing (NGS) 12 revealed that the endometrial microbiome comprises low-biomass microbiota; moreover, it contains fewer bacteria than the vaginal microbiome. 13,14 Multiple studies have demonstrated that ART outcomes are affected by altered microbiota in the vagina, [15][16][17][18][19] uterine cervix, 15,20,21 and endometrium. 12,22 The presence of bacterial pathogens associated with chronic endometritis (CE) induces persistent inflammation of the endometrial mucosa, thereby impairing endometrial receptivity through alterations of decidualization and various cellular mechanisms. 23,24 A recent multicenter prospective study showed that the presence of pathogenic bacteria in the endometrium, together with the depletion of Lactobacillus, was associated with implantation failure. 25 EndomeTRIO (©Igenomix, Valencia, Spain) is a triad of NGSbased tests including ERA, endometrial microbiome metagenomic analysis (EMMA), and analysis of infectious chronic endometritis (ALICE). EMMA is used to evaluate a patient's endometrial microbiota, and ALICE is performed to identify the pathogenic bacteria associated with CE. The EndomeTRIO has simplified endometrial receptivity testing for clinicians and provides additional information that may help develop a therapeutic strategy for ART. However, the mechanisms responsible for differences in receptive status timing are poorly understood because patient confidentiality considerations have hindered the standardization of algorithms used to predict the WOI with these transcriptomes. Here, we conducted a cross-sectional study to identify factors that may affect endometrial receptivity array results. We hypothesized that age-related changes in the endometrial microbiota were involved in the development of endometrial receptivity.

| Study population
We enrolled infertile patients who were planning to undergo FET at the blastocyst stage and who underwent their first EndomeTRIO test at our clinic during the period from August 2021 to July 2022.
All eligible participants had experienced at least 1 unsuccessful embryo transfer (Gardner's grade of 4BB or higher), had at least one remaining frozen blastocyst (Gardner's grade of 4BB or higher), and were given detailed information about the EndomeTRIO test prior to FET. All patients made the decision regarding whether to undergo testing, and informed consent for clinical testing and for inclusion in the study was obtained from all patients. Patients who had used antibiotics, probiotics, or prebiotics within 1 month were excluded from the study.

| Endometrial preparation for testing
The EndomeTRIO test was performed in a programmed hormone replacement cycle, in accordance with the routine FET program at our clinic. Endometrial preparation with transdermal estradiol tape

| Endometrial biopsy
The EndomeTRIO test was performed approximately 108 h after the initial administration of progesterone. After the uterine cervix had been washed with saline solution and the external cervical os had been wiped using a clean cotton ball, an endometrial biopsy was performed using a Pipelle® (Laboratoire CCD). Aspirated tissues (excluding mucus and blood) were placed into a cryotube containing RNAlater (Qiagen), then stored and shipped at 4°C for EndomeTRIO testing, in accordance with the manufacturer's protocol. 1

| Endometrial receptivity analysis
Endometrial tissue was lysed and homogenized with TissueLyser II (Qiagen), and the supernatant was recovered via centrifugation. The patient's RNA was extracted from the supernatant using QIAsymphony SP and QIAsymphony RNA kits (Qiagen), then subjected to reverse transcription to produce cDNA, which was used for library preparation with Ion Torrent (Thermo Fisher Scientific). The expression patterns of 248 genes associated with endometrial receptivity were analyzed using an Ion S5 NGS system (Thermo Fisher Scientific).

| Endometrial microbiome metagenomic analysis
A portion of each specimen subjected to ERA was also tested using EMMA and ALICE. Briefly, 25 mg of endometrial tissue were treated with proteinase K at 56°C for 3 h, then separated and lysed with ATL buffer (Qiagen). Subsequently, the tissues were disrupted with a TissueLyser LT (Qiagen) for 5 min at 50 Hz using stainless steel beads. Bacterial nucleic acids from the sample were purified

| Interpretation of the EndomeTRIO test
The ERA results were interpreted as pre-receptive (profile ≥24 h earlier than the WOI), receptive (profile timed to the WOI), or post-receptive (profile later than the WOI). The EMMA results were interpreted as pattern 1 (normal microbiome with  Odds ratios and 95% confidence intervals related to each variable were calculated. The Wald test was used to assess the overall association. All statistical analyses were conducted using JMP software v. 15.2.1 (SAS Institute Inc.). All tests were two-tailed, and p < 0.05 was considered statistically significant.

| RE SULTS
In this study, we included a total of 185 EndomeTRIO tests for Pre-receptive ERA groups, BMI and serum progesterone levels at initial progesterone administration showed no significant differences. However, patient age was significantly greater in the Prereceptive ERA group, and the distribution of the EMMA results was significantly different; the Pre-receptive ERA group showed a decrease in pattern 1 and an increase in pattern 5 (Table 1). Age was associated with a significant decrease in receptivity according to ERA results and a significant decrease in pattern 1 according to EMMA (Figure 1). The EMMA results were significantly associated with the ERA results. Patients with pattern 1 had a higher rate of receptive ERA results and a lower rate of pre-receptive ERA results; these rates were reversed in patients with pattern 5 ( Figure 2). Multivariable logistic regression analysis revealed that patient age and EMMA results were both significant independent predictive factors for pre-receptive ERA results (Table 2).

| DISCUSS ION
In this observational study, the incidence of pre-receptive endometrium significantly increased with age. Pre-receptive endometrium was also significantly associated with a decreased proportion of Lactobacillus-dominant microbiota and with the presence of dysbiotic microbiota in the endometrium. In particular, women who had a microbiome with ultralow biomass were likely to have pre-receptive endometrium. Both aging and dysbiotic microbiota were independent predictive factors for WOI displacement. After adjustment for other factors, pattern 5 in the EMMA test was most strongly associated with the odds of pre-receptive ERA results. We estimated that the odds of pre-receptive ERA results were 3.8-fold higher for patients with pattern 5 on EMMA than for patients with pattern 1 on EMMA.

F I G U R E 1
Age-related increase in pre-receptive endometrium and decrease in receptive endometrium, interpreted using endometrial receptivity analysis (ERA). The horizontal axis represents patient age, and the vertical axis represents the proportion of endometrial receptivity interpreted using ERA. p = 0.0018 in logistic regression analysis with the Wald test.

F I G U R E 2 Age-related increase in endometrium with ultralow biomass microbiome and decrease in endometrium with
Lactobacillus-dominant microbiota, interpreted using endometrial microbiome metagenomic analysis (EMMA). The horizontal axis represents patient age, and the vertical axis represents the proportions of endometrial microbiota patterns interpreted using EMMA. Numbers 1-5 in the graph represent patterns 1-5. p = 0.0317 in logistic regression analysis with the Wald test.

| The timing of endometrial biopsy
ERA must be conducted with caution in a strictly standardized setting because the results can be affected by various factors. The recommended timing of an endometrial biopsy for ERA testing is 120 h after progesterone administration. 27 However, the timing of endometrial biopsy can slightly vary from 120 h after initial progesterone administration; it is usually performed after 5 days (P + 5).
Although an earlier or later biopsy decreases the proportion of patients with receptive ERA results, the diagnostic algorithm for ERA can predict the WOI with high accuracy. 26 Although endometrial biopsy 108 h after initial progesterone administration did not deviate from recommendations, we guess that biopsy collection 12 h earlier may help expose the effects of age and endometrial microbiota on endometrial receptivity, leading to an increase in the proportion of patients with pre-receptive ERA results. Although the timing of the biopsy is important, there is a greater need to confirm that no endogenous progesterone exposure occurred immediately before the administration of exogenous progesterone; activation of progesterone receptors can trigger the initial development of endometrial receptivity. 2 Endometrial receptivity is mainly regulated by ovarian steroids throughout the cycle. A recent systematic review and meta-analysis did not reveal a significant change in the rate of pregnancy after in vitro fertilization cycles using ERA. 28 However, all studies included in that metaanalysis were retrospective and did not measure endogenous progesterone in ERA cycles; half of the studies included natural cycle FETs. It is difficult to accurately determine the timing of progesterone secretion in the natural cycle solely by using urinary luteinizing hormone tests. 29 We believe that ERA and subsequent FETs to evaluate the efficacy of ERA should be performed in hormone replacement cycles.

| Age-related changes of endometrial receptivity
The timing of WOI is unique to each individual and can persist for up to 40 months after it has been determined. 4 Although the mechanism of age-related changes in endometrial receptivity is unclear, 30 a recent endometrial transcriptomic data analysis revealed that age-related changes begin at the age of 35 years; these changes include upregulation of genes involved in ciliary processes as well as growth factor dysregulation. 31 Our results are consistent with an age-related delay in the development of receptive endometrium. Although obesity is reportedly associated with significant endometrial transcriptomic differences relative to non-obesity, 32 the effect of increased BMI was not apparent in our study, probably because participants were limited to Japanese women, among whom obesity is less common. 33

| Microbiome-related changes of endometrial receptivity
The use of the EndomeTRIO test for simultaneous assessment of endometrial receptivity and endometrial microbiota is less invasive and less costly for patients. We found that microbiota with ultralow biomass (EMMA pattern 5) have detrimental effects on endometrial receptivity. According to a recent systematic review, 34   There has been considerable attention toward the impact of the gut microbiome on a variety of diseases. 35 The gut microbiota can maintain intestinal epithelial homeostasis and promote health.
The surface macromolecules or metabolites of microbes, including Lactobacillus, interact with host cells to produce various cytokines and chemokines that alleviate inflammation and enhance epithelial function. The bacterial composition within each organ system is influenced by individual factors including age, 36 genetics, ethnicity, diet, probiotics, prebiotics, lifestyle, and exposure to antibiotics or other chemicals. 37 Nevertheless, the roles of endometrial microbiota in reproduction and the roles of probiotics in infertility management have not been extensively studied. The endometrial microbiota varies among individuals. Aging is reportedly accompanied by a decrease in the alpha diversity of the endometrial microbiome. 38 The phase of the menstrual cycle may also have a slight influence on the endometrial microbiota; a difference between the follicular and luteal phases has been reported 38 ; a significant decrease in diversity and evenness, occurring around ovulation and continuing into the secretory phase, has also been reported. 39 Moreover, the endometrial microbiota may be altered by exogenous hormones, such as the hormones used in ovarian stimulation, progesterone supplementation, 14

| Conclusions
In this study, we found that aging was accompanied by a decrease in Lactobacillus-dominant microbiota; both aging and endometrial microbiota with ultralow biomass were significantly associated with pre-receptive endometrium. These findings suggest that the quantity (rather than the proportion) of Lactobacillus in the endometrium is involved in the development of endometrial receptivity.
A limitation of the present study is that it constituted a small-scale cross-sectional study at a single private clinic. Nevertheless, these findings are robust because the study was conducted in a strictly standardized setting where endogenous progesterone exposure was excluded. Our findings can assist clinicians in analyses of endometrial receptivity and endometrial microbiota, which may improve therapeutic outcomes in ART. Additional prospective cohort studies are necessary to evaluate the effects of probiotics on endometrial receptivity array results (based on endometrial microbiota composition) and reproductive outcomes. Moreover, extensive research is needed to expand the current understanding of molecular and pathophysiological interactions between microbiota, particularly Lactobacillus, and endometrial receptivity.

ACK N OWLED G M ENTS
We thank Analisa Avila, MPH, ELS, and Ryan Chastain-Gross, PhD, of Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. There was no funding received for this project.

CO N FLI C T O F I NTE R E S T S TATE M E NT
Human rights statements and informed consent: All procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all participants included in the study. The study protocol was approved by the Ethics Committee of our clinic. The authors declare no conflicts of interest for this article. We certify that no person other than the authors has made substantial contributions to the work.